Abstract
The acquisition of the multidrug resistance phenotype in human tumours is associated with an overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with a 190 kDa membrane ATP-binding protein encoded by a multidrug resistance-associated protein (MRP) gene. Human bladder cancer is a highly malignant neoplasm which is refractory to anti-cancer chemotherapy. In order to understand the mechanism underlying multidrug resistance in bladder cancer, we established three doxorubicin-resistant cell lines, T24/ADM-1, T24/ADM-2 and KK47/ADM, and one vincristine-resistant cell line, T24/VCR, from human bladder cancer T24 and KK47 cells respectively. Both T24/ADM-1 and T24/ADM-2 cells which had elevated MRP mRNA levels showed both a cross-resistance to etoposide and a decreased intracellular accumulation of etoposide. T24/VCR cells which had elevated levels of MDR1 mRNA and P-glycoprotein but not of MRP mRNA, showed cross-resistance to doxorubicin. On the other hand, KK47/ADM cells, which had elevated levels of both MRP and MDR1 mRNA and a decreased level of topoisomerase II mRNA, were found to be cross-resistant to etoposide, vincristine and a camptothecin derivative, CPT-11. Our present study demonstrates a concomitant induction of increased levels of MRP mRNA, decreased levels of topoisomerase II mRNA and decreased drug accumulation during development of multidrug resistance in human bladder cancer cells. The enhanced expression of the MRP gene is herein discussed in a possible correlation with the decreased expression of the topoisomerase II gene.
Highlights
The overexpression of membrane P-glycoprotein (P-gp) with Mr of 170 kDa, encoded by the human multidrug resistance 1 (MDRl) gene, is often associated with the acquisition of the multidrug resistance phenotype (Bradley et al, 1988; Gottesman and Pastan, 1988)
MDR1 and multidrug resistance-associated protein (MRP) are both members of the ATP-binding cassette (ABC) superfamily transport system described by Hyde et al (1990), but have very little sequence homology with each other outside the nucleotide-binding domains (Cole et al, 1992)
We examined whether overexpression of MRP is associated with acquired multidrug resistance in human bladder cancer cells
Summary
Both KK47 (Taya et al, 1977) and T24 (Bubenik et al, 1973) were established from transitional cell carcinoma of the bladder and were used as parental cell lines from which multidrug-resistant cells were developed. A vincristine-resistant cell line (T24/ VCR) was established according to the same procedure. These drug-resistant cell lines, KK47/ADM, T24/VCR, T24/. Doubling times of T24/ADM-1, T24/ADM-2 and T24/VCR were 28.0-35.0 h; the doubling time of the parental T24 cells was 25.0 h This finding indicated that the growth rate of each resistant cell line did not differ greatly from that of each parental counterpart. Medium was replaced with 200 fl of buffer (serum-free MEM and 20 mM Hepes, pH 7.5) containing [3Hjetoposide (1 gM, 1 tCi ml- 1) and [3H]vincristine (22 iLM, 0.13 Ci ml-'), and the cells were incubated at 37°C as described previously (Matsuo et al, 1990; Takano et al, 1991; Abe et al, 1994). The cell lysates were mixed thoroughly with 4 ml of Scintisol EX-H (Wako Chemicals, Osaka, Japan) and the radioactivity was determined
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